Abstract Alpha-synuclein (ASYN) is believed to play an important role in the pathology of several neurodegenerative conditions, including some forms of Parkinson's disease, Dementia with Lewy Bodies, and Multiple Systems Atrophy. The most recent hypothesis is that the ASYN proteins (particularly genetic variants such as A53T that are prone to misfolding) are cleaved by the lysosomal enzyme, asparagine endopeptidase (AEP), to generate peptides that promote ASYN aggregation and initiate programmed cell death (PCD) in vulnerable neurons [Zhang et al 2017]. Inhibition of AEP, and of ASYN aggregation, are therefore prime molecular targets for pharmacotherapy of these ?synucleinopathies? [see Brundin et al 2017], and maybe other neurodegenerative diseases. Chemical synthesis has yielded few leads, and this proposal will use a novel plant biotech platform to generate active compounds. In this approach, plant root cells are transformed by expression of human neuronal proteins to make them susceptible to a specific mechanism of neuronal toxicity. Mutants of these transgenic plant cells are then selected for survival when exposed to this neurotoxic mechanism. This mutagenesis and survival selection ?evolves? plant secondary metabolism toward biosynthesis of metabolites that inhibit the neurotoxic mechanism. Proof of concept used expression of the human dopamine transporter (hDAT) in plant cells. This makes the plant cells highly susceptible to cytotoxicity induced by the dopaminergic neurotoxin, MPP+, which is accumulated by the hDAT. When transgenic (hDAT) mutants are selected for survival in MPP+ the resulting sub-population includes many individuals that overproduce known or novel metabolites that inhibit the hDAT, and/or the cytotoxic mechanisms of MPP+ [Brown et al, 2017]. As regards the AEP/ASYN mechanism of neurotoxicity, plant cells naturally contain a homolog to lysosomal AEP, which, like the human neuronal AEP, is linked to PCD [Hatsugai et al, 2015]. Metabolites that regulate this process almost certainly exist in plants, and so mutant plant cells that survive AEP activation should include many that overproduce natural inhibitors of plant (p)AEP. Because plant and human (h)AEP are homologs, these metabolites are potential neuroprotective leads. However, plant cells do not naturally contain homologs of human (h)ASYN so, to mimic the proposed mechanism of synucleinopathies we will create transgenic plant cells that express the hASYN variant A53T. Because pAEP has similar substrate specificity to hAEP, and because the peptides produced by hASYN-A53T cleavage are highly cytotoxic, these transgenic (hASYN- A53T) plant cells should show increased susceptibility to PCD when pAEP is activated (to be established in Phase I). Consequently transgenic (hASYN-A53T) mutants that survive AEP activation should include many individual clones in which inhibitors of pAEP, or of peptide-induced hASYN aggregation, are over-produced. In addition to ?natural? metabolites, mutation and selection should ?evolve? plant secondary metabolism toward novel metabolites with greater inhibitory activity. This will be established in Phase II using conventional in vitro screens, identifying active metabolites by assay-guided fractionation. The objective is to develop these active plant metabolites as therapeutic agents, or leads for synthetic modification, by the applicants in partnership with a major pharmaceutical company. The application of the proprietary biotechnology to these important targets will also strengthen its commercial importance as a plant drug discovery platform.